5-Substituted-3-(2-aminoethylthio)-6-(1-hydroxyethyl)-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid

ABSTRACT

Antibiotic 5-substituted-3-(2-aminoethylthio)-6-(1-hydroxyethyl)-7-oxo-1-azabicyc lo[3.2.0]hept-2-ene-2-carboxylic acids (I) are disclosed:    &lt;IMAGE&gt; I  wherein R is alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl or heteroaryl.

BACKGROUND OF THE INVENTION

This invention relates to antibiotic5-substituted-3-(2-aminoethylthio)-6-(1-hydroxyethyl)-7-oxo-1-azabicyclo(3.2.0)hept-2-ene-2-carboxylicacids (I): ##STR2## Wherein R is substituted or unsubstituted: alkylhaving 1-6 carbon atoms, trifluoromethyl, phenyl, phenylalkyl having7-12 carbon atoms, cycloalkyl having 3-6 carbon atoms, cycloalkylalkylhaving 3-6 ring carbon atoms, and 1-6 carbon atoms in the alkyl moiety,and heteroaryl having 4-6 atoms in the ring, one or more of which isselected from O, N or S; and wherein the substituent or substituents onthe foregoing radicals are selected from: OH, COOH, Cl, F, Br, NH₂,alkyl and alkoxyl having 1-3 carbon atoms.

This invention also relates to the pharmaceutically acceptable salt andester derivatives of I; pharmaceutical compositions comprising I andsuch derivatives; and to methods of treatment comprising administeringsuch compounds and compositions when an antibiotic effect is indicated.

There is a continuing need for new antibiotics, for unfortunately thereis no static effectiveness of a given antibiotic because continued widescale usage of any such antibiotic selectively gives rise to resistantstrains of pathogens. In addition, the known antibiotics suffer from thedisadvantage of being effective only against certain types ofmicroorganisms. Accordingly, the search for new antibiotics continues.

Unexpectedly, it has been found that the compounds of the presentinvention are broad spectrum antibiotics, which are useful in animal andhuman therapy and in inanimate systems.

Thus, it is an object of the present invention to provide a novel classof antibiotics which are 5-substituted analogues of the antibioticthienamycin. These antibiotics are active against a broad range ofpathogens which representatively include both gram positive bacteriasuch as S. aureus, Strep. pyogenes and B. subtilis and gram negativebacteria such as E. coli, Proteus morganii, Serratia, Pseudomonas andKlebsiella. Further objects of this invention are to provide chemicalprocesses for the preparation of such antibiotics and their non-toxicpharmaceutically acceptable salts; pharmaceutical compositionscomprising such antibiotics; and to provide methods of treatmentcomprising administering such antibiotics and compositions when anantibiotic effect is indicated.

The compounds of the present invention (I, above) are convenientlyprepared by the following scheme: ##STR3##

In words relative to the above diagram, the4-substituted-4-(2-substituted-vinyl)azetidine-2-one, 4, startingmaterial is prepared by reacting an R¹ -oxy-3-substituted-butadiene, 1,with chlorosulfonylisocyanate 2. The reaction is conducted withoutsolvent or may be run in solvent such as diethyl ether, ethyl acetate,chloroform, methylene chloride, or the like, at a temperature of from-78° C. to 25° C. for from a few minutes to 1 hour to provide 3. Theradical R¹ is an easily removable acyl blocking group such as analkanoyl or aralkanoyl which bears no functional group or groups whichmight interfere with the desired course of reaction (1+2→3→4).Intermediate species 3 is converted to the sulfinamide by reductionwhich is then hydrolyzed to 4 at pH 6-8. Typically the reaction solutioncomprising 3 is contacted (5-30 minutes) with an aqueous solution (at0°-25° C.) of a reducing agent such as sodium sulfite, thiophenol, orthe like, at pH 6-8 to provide 4.

The reaction 4→5 is a reduction, and is preferably achieved byhydrogenation in a solvent such as ethyl acetate ether, dioxane,tetrahydrofuran (THF), ethanol or the like at 0° to 25° C. for from 5minutes to 2 hours under 1 to 10 atmospheres of hydrogen in the presenceof a hydrogenation catalyst such as platinum metal or oxide thereof suchas 10% Pd/C or the like.

The de-blocking reaction 5→6 is usually desirable when R¹ is acyl topermit the later alkylation, 7→8. The preferred de-blocking procedure isby alcoholysis wherein the solvent is a lower alkanol such as methanol,ethanol or the like in the presence of the corresponding alkali metalalkoxide, such as sodium methoxide. Typically, the reaction is conductedfor from 5 minutes to 1 hour at a temperature of from -10° to 25° C.

Blocking groups R³ and R² are established (6→7) to provide a suitablyprotected species for alkylation (7→8). There is no criticality in thechoice of blocking groups, provided only that they do not interfere withthe intended alkylation. R³ may be hydrogen, a triorganosilyl group suchas trimethylsilyl or the like, or a cyclic ether such as2-tetrahydropyranyl; R² may also be a cyclic ether such as2-tetrahydropyranyl; alternatively R³ and R² may be joined together toform protected species such as 7a: ##STR4## For example, species such as7a are conveniently prepared by treating 6 with 2,2-dimethoxypropane inthe presence of a catalyst such as boron trifluoride etherate, toluenesulphonic acid, or the like in a solvent such as methylene chloride,ether, chloroform, dioxane or the like at a temperature of from -10° C.to 35° C. for from a few minutes to 1 hour.

The alkylation (7→8) is preferably conducted by treating 7 with a strongbase such as lithium diisopropylamide, sodium amide, potassium hydrideor the like in a solvent such as THF, glyme, ether, dimethylformamide(DMF), dimethylsulfoxide (DMSO) or the like at a temperature of from-78° C. to 0° C. The resulting anion is then treated with excessacetaldehyde to provide 8.

The reaction 8→9 establishes the blocking group R⁴ and is typicallyaccomplished by treating 8 with a base such as an alkali metalhydroxide, lithium diisopropylamide,4-dimethylaminopyridine, orn-butyllithium in a solvent such as ether, THF, dioxane, DMF, DMSO orthe like, followed by treatment with an acyl halide of choice such asalkanoyl, aralkanoyl or nuclear substituted aralkanoyl, or alkyl, arylor aralkyl, substituted aralkyl or substituted aryl haloformate such asp-nitrobenzylchloroformate, o-nitrobenzylchloroformate, or the like, ata temperature of from -78° C. to 25° C. for from 1-24 hours.

The de-blocking reaction 9→10 is typically conducted by acid hydrolysissuch as aqueous acetic acid at a temperature of from 25° C. to 75° C.for from 5 minutes to 3 hours.

The aldehyde intermediate 11 is prepared by treating 10 with anoxidizing agent such as CrO₃.2(pyridine) in CH₃ CN, 1:1 mixture ofdimethylsulfoxide and acetic anhydride, cyclohexylcarbodiimide in DMSOor the like at a temperature of from 0°-25° C. for from 5 minutes to 1hour. The resulting species 11 in a solvent such as acetonitrile,methylene chloride, chloroform or the like at a temperature of from -10°to 25° C. is treated with an excess of N-blocked cysteamine, HSCH₂ --CH₂NHR⁵, in the presence of an acid catalyst such as boron trifluorideetherate, toluene sulphonic acid or the like to provide 12. Typically,the reaction requires from 1 to 60 minutes.

There is no criticality as to the identity of the N-protecting group,R⁵, or the cysteamine reagent and suitable groups arep-nitrobenzyloxycarbonyl, o-nitrobenzyloxycarbonyl, t-butyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, phthaloyl, or the like.

The vinyl sulphide 14 is obtained via intermediate 13 by treating 12with a halogen such as chlorine or bromine (X=Cl or Br) in a solventsuch as ether, methylene chloride, tetrahydrofuran, glyme or the like ata temperature of from -78° to 30° C. for from 1 to 30 minutes, followedimmediately by treating with an olefin such as cyclohexene, isobutylene,or the like in the presence of base such as triethylamine, DBU, sodiumhydride, or the like in a solvent such as DMF, glyme, THF, HMPA. Thesolution is held at -20° to 25° C. for from 1 to 8 hours to yield 14.

The vinyl sulphide species 14 is reacted with a diester of oxomalonicacid (or its monohydrate) to provide 15. There is no criticality as tothe identity of the ester moiety, R⁶, of the oxomalonic acid. R⁶ may bea conventional, easily removable blocking group or it may be apharmaceutically acceptable ester moiety. Suitable ester radicals R⁶ arep-nitrobenzyl, benzyl, o-nitrobenzyl, t-butyl, 2,2,2-trichloroethyl. Thereaction 14→15 is typically conducted in a high boiling organic solventsuch as benzene, toluene, cyclohexane, halo aromatic or the like at atemperature of from about 50° C. to reflux for from 0.5 to 6 hours.

The halogenation reaction 15→16 is typically conducted in a solvent suchas THF, glyme, ether, methylene chloride, chloroform or the like in thepresence of a halogenating agent such as thionyl chloride, phosphorouspentachloride or the like in the presence of base such as pyridine at atemperature of from -20° to 25° C. for from 5 minutes to 3 hours. Theselective reduction of 15→17 via 16 is completed by treating 16 withtributylphosphine, triphenylphosphine or the like in aqueous DMF orsimilar aqueous systems involving dioxane, THF, glyme, DMSO, or acetonein the presence of K₂ HPO₄ at a temperature of from about 0°-50° C. forfrom 10 minutes to 5 hours.

Species 17 is halogenated by the previous procedure (12→13), butomitting the addition of the cyclohexene or other olefin, to provide thedihalo species 18. Species 18 is treated with a base such astriethylamine, sodium hydride or potassium hydride in a solvent such asDMF, acetonitrile, methylene chloride, chloroform, glyme or the like ata temperature of from about -78° to 25° C. for 1 to 5 hours to provide19. Species 19 is converted to 20 on treatment with a strong base suchas 1,5-diazabicyclo[5.4.0]undec-5-ene (DBU),1,5-diazabicyclo[3.4.0]non-5-ene(DBN), or the like in a solvent such asDMSO, acetone, chloroform, DMF, THF, glyme or the like or on treatmentwith AgF in pyridine at a temperature of from 0°-40° C. for from 1/4 to24 hours. The reaction 20→21 is conducted by treating 20 with anaromatic base such as pyridine, s-collidine or lutidine, in the presenceof a displacing agent such as lithium iodide, lithium bromide, sodiumbromide, or the like at a temperature of from about 80°-150° C. for from15 minutes to 2 hours. An aqueous work up of the resulting reactionmixture provides 21. Isomerization of the double bond 21→22 isaccomplished by treating 21 in a solvent such as DMF, DMSO, ethyl ether,THF, glyme, methylene chloride with a strong base such asdiisopropylamine, DBU, DBN, or the like at a temperature of from 0° toabout 25° C. for from a few minutes to 2 hours or until equilibrium hasbeen established as determined by examination of sample aliquots byultraviolet absorption or by thin layer chromatography. The finalreaction 22→I (by hydrogenolysis of the blocking groups) is accomplishedby treating 22 in a solvent such as dioxane, ethanol, THF or the like oran aqueous mixture thereof in the presence of a Platinum metal catalystsuch as Pd/C under a hydrogen pressure of from 1-4 atmospheres for from0.5 to 8 hours at a temperature of from about 0°-25° C.

The above-described total synthesis may also advantageously start with a4-substituted-4-vinyl azetidinone (23), below rather than the enolacylate azetidinone (4, above). The following scheme illustrates this4-substituted-4-vinyl-azetidinone embodiment of the present invention;notice that it ties into the above scheme at species 14. ##STR5##

In words relative to the above reaction diagram, the4-substituted-4-vinyl azetidinone 23 is silylated to provide the N-silylspecies 24. The groups R' on the silyl radical are loweralkyl havingfrom 1-6 carbon atoms especially preferred triorganosilyl groups aretrimethylsilyl and t-butyl-dimethylsilyl. Typically the silylation(23-24) is achieved by treating 23 in a solvent such as DMF, DMSO, HMPAor the like with the silylating agent of choice, dimethyl t-butylsilylchloride, and a base such as Et₃ N, pyridine, N,N-dimethylaniline andthe like at a temperature of from -10° to 30° C. for from 1 to 8 hours.Species 24 is alkylated to form 25 by treatment with acetaldehyde in thepresence of base. This reaction 24→25 is conducted exactly as describedabove for the alkylation 7→8. The O-protecting group is established inthe reaction 25→26. The protecting group R⁴ is as previously defined andthe reaction 25→26 is exactly analogous to the above described reaction8→9. The removal of the N-triorganosilyl group is accomplished inreaction 26→27 by mild acid catalyzed solvolysis. The halo sulfidespecies 28 is obtained from 27 by treating 27 in a solvent such asmethylene chloride, THF, glyme, or the like with the reagent XSCH₂ CH₂NHR⁵ wherein R⁵ has previously been defined and X is halogen such aschloro or bromo at a temperature of from -50° C. to 50° C. for from 1 to16 hours. The final sulfide intermediate 14, which is common to theabove illustrated scheme of total synthesis is obtained from 28 byelimination of HX on treatment of 28 with a base such as1,5-diazabicyclo (5.4.0)undec-5-ene (DBU),1,5-diazabicyclo[4.3.0]non-5-ene, (DBN), 1,4-diazabicyclo[2.2.2]octane,(DABCO), or silver fluoride in a solvent such as DMSO, pyridine, DMF,HMPA or the like at a temperature of from -20° to 50° C. for from 1/4 to16 hours.

The products of this invention (I) form a wide variety ofpharmacologically acceptable salts with inorganic and organic bases;these include, for example, metal salts derived from alkali or alkalineearth metal hydroxides, carbonates or bicarbonates and salts derivedfrom primary, secondary or tertiary amines such as monoalkylamines,dialkylamines, trialkylamines, loweralkanolamines,di-loweralkanolamines, lower alkylenediamines, N,N-diaralkyl loweralkylenediamines, aralkylamines, amino substituted lower alkanols,N,N-di-lower alkylamino substituted lower alkanols, amino-, polyamino-and guanidino-substituted lower alkanoic acids and nitrogen-containingheterocyclic amines. Representative examples include salts derived fromsodium hydroxide, sodium carbonate, sodium bicarbonate, potassiumcarbonate, potassium hydroxide, calcium carbonate, trimethylamine,triethylamine, piperidine, morpholine, quinine, lysine, protamine,arginine, procaine, ethanolamine, morphine, benzylamine,ethylenediamine, N,N'-dibenzylethylenediamine, diethanolamine,piperazine, dimethylaminoethanol, 2-amino-2-methyl-1-propanol,theophylline, N-methylglucamine and the like. Acid addition salts, e.g.,with hydrochloric, tartaric, hydrobromic, sulfuric nitric,toluene-p-sulphonic and methane sulphonic acids may also be employed.

The salts can be mono-salts such as the monosodium salt obtained bytreating one equivalent of sodium hydroxide with one equivalent of theproduct (I), also mixed di-salts. Such salts may be obtained by treatingone equivalent of a base having a divalent cation, such as calciumhydroxide, with one equivalent of the product (I). The salts of thisinvention are pharmacologically acceptable non-toxic derivatives whichcan be used as the active ingredient in suitable unit-dosagepharmaceutical forms. Also, they may be combined with other drugs toprovide compositions having a broad spectrum of activity.

The compounds of the present invention are valuable antimicrobialsubstances which are active against various gram-positive andgram-negative pathogens. Thus the free acid, free base, and especiallythe salts thereof such as amine and metal salts, particularly the alkalimetal and alkaline earth metal salts, are useful bactericides and can beused for removing susceptible pathogens from dental and medicalequipment, for separating microorganisms, and for therapeutic use inhumans and animals. For this latter purpose pharmacologically acceptablesalts with inorganic and organic bases such as those known in the artand used for the administration of penicillins and cephalosporins can beutilized. For example, salts such as alkali metal and alkaline earthmetal salts, and primary, secondary and tertiary amine salts can be usedfor this purpose. These salts can be combined with pharmaceuticallyacceptable liquid and solid vehicles to form suitable dosage unit formssuch as pills, tablets, capsules suppositories, syrups, elixirs and thelike which can be prepared in accordance with procedures well known inthis art.

The novel compounds are valuable antibiotics active against variousgram-positive and gram-negative bacteria, and accordingly, find utilityin human and veterinary medicine. The compounds of this invention cantherefore be used as antibacterial drugs for treating infections causedby gram-positive or gram-negative bacteria, for example againstStaphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Bacillussubtilis, Salmonella typhosa, Pseudomonas and Bacterium proteus. Theantibacterials of the invention may further be utilized as additives toanimal feedingstuffs, for preserving foodstuffs and disinfectants. Forexample, they may be employed in aqueous compositions in concentrationsranging from 0.1 to 100 parts of antibiotic per million parts ofsolution in order to destroy and inhibit the growth of harmful bacteriaon medical and dental equipment and as bactericides in industrialapplication, for example in waterbased paints and in the white water ofpaper mills to inhibit the growth of harmful bacteria.

The products of this invention may be used alone or in combination as anactive ingredient in any one of a variety of pharmaceuticalpreparations. These antibiotics and their corresponding salts may beemployed in capsule form or as tablets, powders or liquid solutions oras suspensions or elixirs. They may be administered orally,intravenously or intramuscularly.

The compositions are preparably presented in a form suitable forabsorption by the gastro-intestinal tract. Tablets and capsules for oraladministration may be in unit dose presentation form, and may containconventional excipients such as binding agents, for example syrup,acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillersfor example, lactose, sugar, maize-starch, calcium phosphate, sorbitolor glycine; lubricants, for example, magnesium stearate, talc,polyethylene glycol, silica; disintegrants, for example, potato starchor acceptable wetting agents such as sodium lauryl sulphate. The tabletsmay be coated according to methods well known in the art. Oral liquidpreparations may be in the form of aqueous or oily suspension, solution,emulsions, syrups, elixirs, etc. or may be presented as a dry product,for reconstitution with water or other suitable vehicles before use.Such liquid preparations may contain conventional additives such assuspending agents, for example, sorbitol syrup, methyl cellulose,glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible oils, forexample almond oil, fractionated coconut oil, oily esters, propyleneglycol, or ethyl alcohol; preservatives, for example methyl or propylp-hydroxybenzoates or sorbic acid. Suppositories will containconventional suppository bases, e.g., cocoa butter of other glyceride.

Compositions for injection may be presented in unit dose form inampules, or in multidose containers with an added perservative. Thecompositions may take such forms as suspensions, solutions, or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form for reconstitution with asuitable vehicle, e.g., sterile, pyrogen-free water, before use.

The compositions may also be prepared in suitable forms for absorptionthrough the mucous membranes of the nose and throat or bronchial tissuesand may conveniently take the form of powder or liquids sprays orinhalants, lozenges, throat paints, etc. For medication of the eyes orears, the preparations may be presented as individual capsules, inliquid or semi-solid form, or may be used as drops etc. Topicalapplications may be formulated in hydrophobic or hydrophilic bases asointments, creams, lotions, paints, powders, etc.

Also, in addition to a carrier, the instant compositions may includeother ingredients such as stabilizers, binders, antioxidants,preservatives, lubricators, suspending agents, viscosity agents orflavoring agents and the like. In addition, there may also be includedin the composition other active ingredients to provide a broaderspectrum of antibiotic activity.

For veterinary medicine the composition may, for example, be formulatedas an intramammary preparation in either long acting or quick-releasebases.

The dosage to be administered depends to a large extent upon thecondition of the subject being treated and the weight of the host, theroute and frequency of adminstration, the parenteral route beingpreferred for generalized infections and the oral route for intestinalinfections. In general, a daily oral dosage consists of from about 5 toabout 600 mg. of active ingredient per kg. of body weight of the subjectin one or more applications per day. A preferred daily dosage for adulthumans lies in the range of from about 15 to 240 mg. of activeingredient per kg. of body weight.

The instant compositions may be administered in several unit dosageforms as, for example, in solid or liquid orally ingestible dosage form.The compositions per unit dosage, whether liquid or solid may containfrom 0.1% to 99% of active material, the preferred range being fromabout 10-60%. The compositions will generally contain from about 15 mg.to about 1500 mg. of the active ingredient; however, in general, it ispreferable to employ a dosage amount in the range of from about 250 mg.of 1000 mg. In parenteral administration the unit dosage is usually thepure compound in a slightly acidified sterile water solution or in theform of a soluble powder intended for solution.

The following examples, illustrate but do not limit the product,process, compositional or method of treatment aspects of the presentinvention.

EXAMPLE 1 Preparation of 4-(2-acetoxyvinyl)-4-methyl-azetidine-2-one##STR6##

A solution of 1.0 ml distilled chlorosulfonylisocyanate (1.65 g; 11.7mmoles) in 2.5 ml anhydrous diethyl ether is cooled under N₂ in a -20°C. bath.

A solution of 1-acetoxy-3-methyl-butadiene (22 mmoles) in 2.5 mlanhydrous ether is similarly cooled under N₂ in a -20° C. bath.

The chlorosulfonylisocyanate solution is added dropwise to theacetoxybutadiene solution by means of a Teflon tube immersed in the CSIsolution and pressurized with N₂. The addition takes 10 minutes, and thereaction is stirred at -20° C. for 0.5 hour.

A solution of 2 g sodium sulfite and 5 g K₂ HPO₄ in 20 ml H₂ O isprepared during the above 0.5 hour reaction time and is cooled in an icebath; 20 ml of ether is added and the mixture is vigorously stirred inan ice bath. At the end of the 30 minute reaction time, the reactionmixture is transferred, again using N₂ pressure and the Teflon tube,from the reaction flask which is maintained in the -20° C. bath, to thevigorously stirred hydrolysis mixture. Rapid dropwise addition iscompleted in 5 minutes. The hydrolysis is allowed to continue for 5additional minutes. The hydrolysis mix has a pH of 6-8, preferably pH 8.

The phases are separated, the ether phase is dried with MgSO₄. Theaqueous phase is extracted three more times with 50 ml portions ofether, each being added to the initial ether/MgSO₄.

The dried extracts are filtered and concentrated under a N₂ stream to 5ml.

A column of 10 g Baker silica gel, packed in ether is prepared, and theether concentrate is applied to the top. Elution with ether affords thetitle compound.

EXAMPLE 2 Preparation of 4-(2-acetoxyethyl)-4-methyl-2-azetidinone##STR7##

A solution of 4-(2-acetoxyvinyl)-4-methyl-2-azetidinone (10.0 g) 200 mlethyl acetate containing 100 mg of 10% Pd/C is hydrogenated on a Parrshaker at 25° C. under 40 psi hydrogen for 15 minutes. The mixture isfiltered through a bed of Supercel and washed with additional ethylacetate. The combined filtrate is evaporated in vacuo to give4-(2-acetoxyethyl)-4-methyl-2-azetidinone.

EXAMPLE 3 Preparation of 4-(2-hydroxyethyl)-4-methyl-2-azetidinone##STR8##

Under nitrogen at 0°, a solution of4-(2-acetoxyethyl)-4-methyl-2-azetidinone (0.014 mole) in 25 mlanhydrous methanol is treated with a solution of sodium methoxide (77mg, 1.4 mmoles) in 5 ml anhydrous methanol. After stirring for 1 hour,the solution is neutralized with glacial acetic acid. Removal of themethanol in vacuo gives crude 4-(2-hydroxyethyl)-4-methyl-2-azetidinoneas an oil. The product is purified by chromatography on silica geleluting with 10% MeOH/CHCl₃ to give the alcohol.

EXAMPLE 4 Preparation of8-oxo-2,2,6-trimethyl-3-oxa-1-azabicylco[4.2.0]octane ##STR9##

A solution of 4-(2-hydroxyethyl)-4-methyl-2-azetidinone (0.016 mole) and2,2-dimethoxypropane (1.69 g, 0.016 mole) in 25 ml anhydrous methylenechloride is treated with boron trifluoride etherate (0.201 ml, 0.002mole) at 25° C. The resulting solution is stirred for ten minutes.Removal of the solvent under reduced pressure gives an oil (2.5 g).Chromatography of the crude product on silica gel using 2:1 ethylacetate/benzene as eluting solvent gives8-oxo-2,2,6-trimethyl-3-oxa-1-azabicyclo[4.2.0]-octane.

EXAMPLE 5 Preparation of8-Oxo-2,2,6-trimethyl-7-(1-hydroxyethyl)-3-oxa-1-azabicyclo[4.2.0]octane##STR10##

To a solution of 1.1 equivalents of freshly prepared lithiumdiisopropylamide in anhydrous tetrahydrofuran under a nitrogenatmosphere at -78° is added a solution of 8-oxo-2,2,6-trimethyl-3-oxa-1-azabicyclo[4.2.0]octane in anhydrous tetrahydrofuran which hasbeen cooled to -78° C. After two minutes, the resulting lithium enolateis treated with excess acetaldehyde. The solution is stirred for 30minutes at -78° and then poured into water. The aqueous phase issaturated with sodium chloride and extracted with ethyl acetate. Thecombined ethyl acetate solutions are dried over magnesium sulfate andfiltered. The filtrate is evaporated under reduced pressure to give thecrude product. Purification by chromatography on silica gel usingethylacetate/benzene gives8-oxo-2,2,6-trimethyl-7-(1-hydroxyethyl)-3-oxa-1-azabicyclo[4.2.0]octane.

EXAMPLE 6 Preparation of8-Oxo-2,2,6-trimethyl-7-(1-p-nitrobenzylcarbonyldioxyethyl)-3-oxa-1-azabicyclo[4.2.0]octane##STR11##

Under anhydrous conditions at 0° C. a solution of8-oxo-2,2,6-trimethyl-7-(1-hydroxyethyl)-3-oxa-1-azabicyclo[4.2.0]octane(60 mg, 0.302 mmole) in 0.6 ml ether is treated with powdered potassiumhydroxide (19 mg, 0.332 mmole). After a period of 15 minutes,p-nitrobenzylchloroformate (65 mg, 0.302 mmole) is added to the reactionmixture. Stirring is continued at 25° C. for an additional 15 hours. Themixture is partitioned between 1 M pH 7 phosphate buffer and more ether.The ether phase is washed with water and brine, dried over magnesiumsulfate and filtered. Evaporation of the filtrate under reduced pressureand purification by preparative thick-layer chromatography on silica geldeveloping with 1:9 ethyl acetate/benzene gives8-oxo-2,2,6-trimethyl-7-(1-p-nitrobenzylcarbonyldioxyethyl)-3-oxa-1-azabicyclo[4.2.0]octane.

EXAMPLE 7 Preparation of3-(1-p-nitrobenzylcarbonyldioxyethyl)-4-(2-hydroxyethyl)-4-methyl-2-azetidinone##STR12##

8-Oxo-3-oxa-2,2,6-trimethyl-7-(1-p-nitrobenzylcarbonyldioxyethyl)-1-azabicyclo[4.2.0]octane(1.0 g) is dissolved in 8 ml acetic acid and 2 ml water and heated at65° C. for 1.25 hours. The acetic acid and water are removed underreduced pressure and the residue is taken up in benzene and evaporatedto give3-(1-p-nitrobenzylcarbonyldioxyethyl)-4-(2-hydroxyethyl)-4-methyl-2-azetidinone.

EXAMPLE 8 Preparation of5-methyl-3-(2-aminoethylthio)-6-(1-hydroxyethyl)-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylicacid ##STR13##

To 6.75 ml anhydrous pyridine (mw=79; ρ=0.982; 83.9 mmole) in 350 mlanhydrous acetonitrile is added 4.05 g anhydrous powdered chromiumtrioxide (mw=100; 40.5 mmole). After stirring at room temperature (25°C.) for 30 minutes, 9.6 g dried Supercell is added and stirring iscontinued for 5 additional minutes. A solution of3-(1-p-nitrobenzylcarbonyldioxyethyl)-4-methyl-4-(2-hydroxyethyl)-2-azetidinone(9.5 mmole) in 30 ml anhydrous acetonitrile is added all at once. Thereaction mixture is stirred under anhydrous conditions at roomtemperature (25° C.) for one hour. Addition of 9.6 g NaHSO₃ is followedby 5 minutes of stirring after which the reaction mixture is filteredthrough a mixed packed bed of 40 g silica gel and 40 g anhydrousmagnesium sulfate. The bed is washed repeatedly with acetonitrile (totalvolume of filtrate˜600 ml). The filtrate is concentrated under a N₂stream to 130 ml total volume. To this solution containing crudealdehyde at 0° C. under N₂ is added 9.64 gp-nitrobenzyloxycarbonylaminoethanethiol (mw=256; 37.7 mmole) asprepared below (Example 8, Step B). To the stirred reaction mixture isadded 8.0 ml boron trifluoride etherate (mw=142; ρ=1.125; 63.4 mmole).After 1.5 hours at 0° C., the reaction mixture is poured into a stirredice-cold mixture of 69 g K₂ HPO₄ -500 ml H₂ O and 700 ml ethyl acetate(EA). The layers are separated and the aqueous one is saturated withNaCl and re-extracted with additional EA. The combination organic layersare washed twice with brine, dried over anhydrous MgSO₄ and filtered.The filtrate is concentrated under a N₂ stream and then pumped on highvacuum to give crude 1.

The material is chromatographed on 450 g silica gel (column height=48cm; diameter=5.5 cm) packed and applied in CHCl₃ and eluted withincreasing percentages of MeOH in CHCl₃ (0-4% MeOH/CHCl₃). Thosefractions containing the desired product are combined, concentratedunder N₂ stream; and pumped on high vacuum to give 1. ##STR14##

To 600 ml diethyl ether (Et₂ O)-75 ml H₂ O in an ice bath with stirringis added 3.2 g cysteamine hydrochloride (mw=114; 28.1 mmole). A solutionof 7.14 g NaHCO₃ (mw=84; 85 mmole) in 75 ml H₂ O is added. The ice bathis removed, and at room temperature a solution of 6.75 gp-nitrobenzylchloroformate (mw=216; 31.3 mmole) in 270 ml Et₂ O is addeddropwise over a period of one hour. After 10 additional minutes, thelayers are separated. The ether layer is extracted with 150 ml 0.25 NHCl, and then with 200 ml brine. Each aqueous layer is then backwashedsuccessively with 100 ml Et₂ O. The combined Et₂ O layers are dried overanhydrous MgSO₄, filtered, and concentrated under a N₂ stream. Thecrystalline residue is slurried in a small amount of ether, filtered,and the pale yellow crystals are dried under high vacuum to give 4.7 gp-nitrobenzyloxycarbonylaminoethanethiol (65% yield).

NMR (CDCl₃): 8.18 (d, J=8 Hz, aromatic protons ortho to nitro), 7.47 (d,J=8 Hz, aromatic protons meta to nitro), 5.27 (--NH--), 5.20 (s, --CH₂-- φ-pNO₂), 3.40 (m, --CH₂ --NH--), 2.67 (m, --CH₂ --SH), 1.35 (t, J=8.5Hz, --SH) in ppm downfield from TMS.

IR (CHCl₃ solution) carbonyl- ˜ 1725 cm⁻¹

M.S.-molecular ion-256, (M-47) at 209, (M-136) at 120, +CH₂ φpNO₂ at136. ##STR15##

To 14.2 ml pentane (dried over 4A Linde molecular sieves) is added 0.5ml Br₂ (mw=160; 9.75 mmole). To 6.02 mmole of 1 in 58 ml tetrahydrofuran(THF) (freshly distilled from lithium aluminum hydride) (LAH) and 65 mlEt₂ O (dried over 3A 1/16" Linde molecular sieves) at 0° C. under N₂with stirring is added dropwise 10 ml of the above 0.66 M Br₂ solution(6.6 mmole). After 10 minutes at 0° C., 0.67 ml cyclohexene (mw=82;ρ=0.81; 6.6 mmole) is added. After 5 minutes at 0° C., 1.7 mltriethylamine (mw=101; ρ=0.729; 12.3 mmole) is added immediatelyfollowed by 40 ml ice-cold dimethylformamide (DMF) (distilled fromanhydrous CaSO₄ at 40 mm and stored over 4A Linde molecular sieves). Theice bath is removed, and stirring is continued for 21/4 hours at roomtemperature. The reaction mixture is poured into a stirred ice-coldmixture of 12.6 ml 1 M KH₂ PO₄ 160 ml H₂ O-500 ml (EA). After separationof the layers, the aqueous one is saturated with sodium chloride andre-extracted with EA. The combined organic layers are extracted oncewith brine, dried over anhydrous MgSO₄, filtered and concentrated underN₂ stream followed by pumping under high vacuum to provide crude 2.

The material is chromatographed on 250 g silica gel (height=45 cm;diameter=4.5 cm) packed and applied in CHCl₃ and eluted with increasingpercentages of MeOH in CHCl₃ (0-3% MeOH/CHCl₃). Those fractionscontaining clean product are combined, concentrated under a N₂ stream,and pumped on high vacuum to give 2. Contaminated fractions arerechromatographed on silica gel using increasing percentages of EA inCHCl₃ (0-25% EA/CHCl₃) to give additional 2. ##STR16##

To a stirred solution of 2.48 g di(p-nitrobenzyl)ketomalonate (fromExample 8, Step E) (mw=388; 6.39 mmole) in 400 ml hot anhydrous tolueneis added a solution of 2 (4.39 mmole) in 20 ml THF (distilled from LAH)and 40 ml anhydrous toluene. After some of the solvent is boiled off,additional anhydrous toluene is added, and the azeodrying process isrepeated three times. The solution is then refluxed under N₂ for 30minutes. Additional toluene is then allowed to boil off yet the volumeis not allowed to diminish to much that precipitation occurs. Totalheating time is approximately 21/2 hours. The clear yellow reactionmixture is removed from the oil bath and placed under a stream of N₂which instantaneously causes clouding. After concentration, the residueis dissolved in CH₂ Cl₂, dried over anhydrous MgSO₄, filtered, andconcentrated under a N₂ stream to give crude 3.

The material is chromatographed on 250 g silica gel packed and appliedin CHCl₃ (height=43 cm; diameter=4.5 cm). Elution with 500 ml 0.5%MeOH/CHCl₃ is followed by continued elution with 1% MeOH/CHCl₃ for theremainder of the chromatography. After the emergence of excess reagent,those fractions containing pure 3 are combined, concentrated under a N₂stream and then on high vacuum to give 3. ##STR17##

A mixture of 100 g p-nitrobenzyl bromide (0.46 mole), 28.6 g malonicacid (0.275 mole) and 750 ml ethanol (EtOH) is stirred and warmed on thesteam bath until solution is achieved. A solution of 33 g KOH (>85%purity; ˜0.6 mole) in 200 ml of water is added carefully with swirling.An additional 200 ml of water is added, and the two-phase system isrefluxed for 1.8 hours. The lighter color homogeneous solution is cooledin ice for 1 hour and the crude product isolated by filtration, washedtwice with a minimum of cold EtOH, and dried by pulling dry N₂ throughthe cake; 33.7 g of solid is obtained. If, during the refluxing stagethe reaction mixture is slowly concentrated to ca. half volume byallowing refluxing solvent to distill off, the crude product yield risesto 77 g. The material is recrystallized from methanol to give puredi-p-nitrobenzyl malonate 1'.

A mixture of 23.4 g of 1', 10 g SeO₂, and 30-40 ml of xylene is stirredin a flask immersed in an oil bath. The bath temperature is raised over1 hour to 130°14 135°. A gradual darkening of the reaction mixture isnoted, and after a total of 4 hours at 130°-135°, most of the insolubleresidue is black Se°. The mixture is cooled, MgSO₄ is added to removethe water, and Celite is added to aid in filtration. The mixture isfiltered through Celite and the cake washed with xylene and a smallportion of EtOAc. Final volume: 60 ml. A 100 g column of Baker SilicaGel is prepared in benzene and 10 ml of filtrate applied, then elutedwith increasing amounts of EtOAc in benzene, 500 ml fractions beingcollected. After one 2% ethyl acetate (EtOAc)/φH, and two 10% EtOAc/φHfractions, the third 10% and first 20% EtOAc/φH provide the bulk of theproduct (˜1.6 g from 10 ml filtrate) as judged by tlc (20% EtOAc/CHCl₃ ;silica gel GF). Recrystallization from benzene, (1 g in ca. 50 mlconcentrated to ˜1/3 volume and "spiked" with 1 ml of H₂ O-saturatedbenzene): provides 0.24 g 2'; mp (117) 121°-122°. [φH=benzene.]##STR18##

A solution of 1.53 mmole of 3 in CH₂ Cl₂ is dried over anhydrous MgSO₄,filtered, concentrated under a N₂ stream, and dried further under highvacuum just prior to the following reaction. To a solution of 3 in 24 mlTHF (freshly distilled from LAH) at -20° C. is added 0.206 ml anhydrouspyridine (mw=79; ρ=0.982; 2.56 mmole). With stirring under N₂, 294 mg offreshly distilled thionyl chloride (mw=119; 2.47 mmole) in 5 ml THF isadded dropwise. The reaction mixture is stirred for 10 minutes at -20°C., then 1/2 hour at 0° C. and finally 1 hour at 25° C. The pyridinehydrochloride is filtered under N₂ and washed with 20 ml THF. Thefiltrate is concentrated under a N₂ stream followed by pumping on highvacuum. The resulting foam is swirled in 25 ml anhydrous THF, and asmall amount of orange-red insoluble material is filtered off under N₂.The filtrate is re-concentrated as above to a foam.

To this freshly prepared chloro compound is added with stirring afreshly shaken suspension of 678 mg tributylphosphine (mw=202; 3.36mmole) in 36.5 ml 9:1 DMF-H₂ O followed by 294 mg K₂ HPO₄ (mw=174; 1.69mmole). The reaction mixture is stirred at 25° C., for 35 minutes. Afterdilution with 120 ml EA and 60 ml brine, the layers are separated, andthe aqueous one is extracted two times with EA. The combined organiclayers are washed one time with brine, dried over anhydrous MgSO₄,filtered and concentrated under a N₂ stream followed by pumping on highvacuum to give crude 4.

The material is chromatographed on 100 g silica gel (height=28.5 cm; d=4cm) packed and applied in CHCl₃ and eluted with 0.5% MeOH in CHCl₃.Those fractions containing clean product are combined, concentratedunder a N₂ stream and then on high vacuum to give 4. Contaminatedfractions are re-chromatographed on silica gel thin layer plates(eluant=50% acetone/hexane; extraction of desired u.v. band with CHCl₃and EA) to provide additional 4. ##STR19##

To 8.5 ml pentane (dried over 4A Linde molecular sieves) is added 0.2 mlBr₂ (mw=160; 3.9 mmole). To 0.746 mmole of 4 in 18 ml THF (freshlydistilled from LAH) and 5.7 ml Et₂ O (dried over 3A 1/16" Lindemolecular sieves) at 0° C. under N₂ with stirring is added dropwise 1.8ml of the above 0.45 M Br₂ solution (0.81 mmole). After 15 minutes at 0°C., 0.42 ml triethylamine (mw=101; ρ=0.729; 3.03 mmole) is added,immediately followed by 10.5 ml ice-cold DMF (distilled from CaSO₄ at 40mm and stored over 4A Linde molecular sieves). The ice-bath is removed,and stirring at room temperature is continued for 2 hours. The reactionmixture is poured into a stirred, ice-cold mixture of 3.1 1 M KH₂ PO₄-70 ml H₂ O-100 ml EA. The layers are separated, and the aqueous one issaturated with NaCl and re-extracted with EA. The combined organiclayers are washed once with brine, dried over anhydrous MgSO₄, andfiltered. The filtrate is concentrated under a N₂ stream and then pumpedon high vacuum to give crude 5.

The material is chromatographed on 60 g silica gel (diameter=2.8 cm)packed and applied in CHCl₃ and is eluted with 100 ml-2% EA/CHCl₃ ; 100ml-4% EA/CHCl₃ and then 5% EA/CHCl₃ for the remainder of thechromatography. The fractions containing pure 5 are combined,concentrated under a N₂ stream, and pumped on high vacuum to give 5.##STR20##

To 29 mg anhydrous silver fluoride (mw=127; 0.23 mmole) is added asolution of 0.14 mmole of 5 in 3.5 ml anhydrous pyridine. The stopperedreaction mixture is stirred at room temperature in the dark for one hourand then poured into 20 ml cold water -30 ml EA. After separation of thelayers, the aqueous one is extracted two times with EA and one time withCHCl₃. Each organic layer is extracted one time with H₂ O and one timewith brine. The combined organic layers are dried over anhydrous MgSO₄,filtered, and concentrated under a N₂ stream followed by pumping on highvacuum to give crude 6.

Preparative thin layer chromatography (eluant=40% acetone/hexane;repeated extraction of desired u.v. band with a large volume of CHCl₃)yields slightly contaminated 6. Re-chromatography on silica using EA inCHCl₃ as an eluting system gives pure 6. ##STR21##

A solution of 0.082 mmole of 6 in 0.9 ml S-collidine (distilled frompowdered KOH˜30 mm pressure) is added to 13.4 mg anhydrous LiI (driedfor few hours at 100° C. over P₂ O₅ under vacuum) (mw=134; 0.1 mmole).With stirring under N₂, the reaction mixture is heated in an oil bath in120° C. After a total of 30 minutes, the reaction mixture is cooled to25° C., diluted with CH₂ Cl₂, and transferred to a round bottom flaskfor concentration under a N₂ stream and then on high vacuum.Partitioning the residue between EA-H₂ O and 1 ml 1 M KH₂ PO₄ isfollowed by extraction of the aqueous layer two additional times with EAand one time with CHCl₃. Each organic layer is then backwashed withbrine. The combined organic layers are dried over anhydrous MgSO₄,filtered, concentrated under a N₂ stream and then on high vacuum to givecrude 7.

Preparative thin layer chromatography on silica gel (plate is eluted twotimes with 40% acetone/hexane; repeated extraction of desired u.v. bandswith large volume of CHCl₃) yields recovered starting material and thedesired product 7. ##STR22##

To 0.064 mmole of 7 in 0.7 ml DMSO (distilled from CaH₂ at 8 mm andstored over 4A Linde molecular sieves) is added 100 μl diisopropylamine(distilled from NaH under N₂ and stored over 4A Linde molecular sieves)(mw=101; ρ=0.722; 0.71 mmole). The stoppered reaction mixture is stirredfor a few minutes and then allowed to stand for 2 hours. The amine andmost of the DMSO are then concentrated off under high vacuum with noexternal heating. The residue is passed quickly through a column ofsilica gel (packed, applied, and eluted with EA) to remove residualDMSO. After concentration under a N₂ stream of all fractions having u.v.absorbance, the material is chromatographed on a thin layer silica gelplate (eluant=50% EA/CHCl₃ ; repeated extraction of desired u.v. bandswith a large volume of chloroform) to give the pure product 8 andunisomerized 7. Starting material is re-submitted to the reactionconditions and isolation procedure two more times to yield additional 8.##STR23##

To ca.5 mg 8 is added 0.60 ml dioxane, 0.05 ml ethanol, 0.35 mldeionized water and 0.01 ml of 1.0 M K₂ HPO₄. To the resultant clearsolution is added 5 mg of 10% Pd/C. The suspension is flushed with N₂,then 5-6 times alternately with 50 psi H₂ and vacuum. Finally, it isshaken under a 50 psi H₂ atmosphere for 30-40 min. After centrifugation,the Pd/C is washed and centrifuged 2-3× with 0.5 ml portions ofdeionized water. The combined centrifugates are extracted 5×1-2 mlether. Residual ether is removed under vacuum and the aqueous solutionapplied to an XAD-2 column (20×140 mm). Fractions of 100 drops (6-7 ml)are collected, with continuous UV monitoring, eluting with deionizedwater. Emergence of strongly UV absorbing material begins aroundfractions 3-5 and is usually complete by fractions 25-30. Earlyfractions are examined by UV to exclude those few deemed too stronglyabsorbing in the 270-280 mμ region. The remaining fractions are combinedand lyophilized. The residue is evaluated by dissolving in 10.0 ml ofdeionized water and measuring the UV absorbtion at 298 mμ.

EXAMPLE 9 Step A Preparation of24[1-(t-butyldimethylsilyl)-4-methyl-4-vinyl-2-azetidinone] ##STR24##

A solution of 23[4-methyl-4-vinyl-2-azetdinone] (11.89 mmoles) andtriethylamine (1.82 ml, 13.08 mmoles) in anhydrous N,N-dimethylformamideis placed under a nitrogen atmosphere, cooled to 0° C. and treated witht-butyldimethylchlorosilane (1.885 g., 12.48 mmoles) resulting in theimmediate appearance of a heavy white precipitate. This mixture isstirred for one hour while gradually warming to room temperature. Themixture is partitioned between 30 ml. methylene chloride and 90 ml cold1 M potassium dihydrogen phosphate. The aqueous phase is extracted with20 ml methylene chloride. The combined organic phases are washed fourtimes with 30 ml portions of water and finally with 50 ml brine. Themethylene chloride solution is dried over anhydrous magnesium sulfateand filtered. The filtrate is evaporated under reduced pressure to give24[1-(t-butyldimethylsilyl)-4-vinyl-4-methyl-2-azetidinone].

Step B Preparation of25[1-(t-butyldimethylsilyl)-3-(1-hydroxyethyl)-4-methyl-4-vinyl-2-azetidinone]##STR25##

To a solution of freshly prepared lithium diisopropylamide (7.82 mmoles)in 36 ml anhydrous tetrahydrofuran under a nitrogen atmosphere at -75°C. is added a solution of24[1-(t-butyldimethylsilyl)-4-vinyl-2-azetidinone] (7.11 mmoles) in 10ml anhydrous THF. The resulting yellow solution of the lithium enolateis, after 16 minutes, treated with acetaldehyde (1.59 ml, 28.4 mmoles).In 10 minutes, the reaction is quenched by adding 30 ml of a saturatedaqueous ammonium chloride solution. This mixture is extracted with 50 mland 25 ml portions of ethyl acetate. The combined ethyl acetatesolutions are washed with 50 ml of brine and dried over anhydrousmagnesium sulfate. The drying agent is removed by filtration and thefiltrate is evaporated in vacuo to give the crude product as a yellowoil. Purification by chromatography on silica gel eluting with 10% ethylacetate/chloroform gives25[1-(t-butyldimethylsilyl)-3-(1-hydroxyethyl)-4-vinyl-4-methyl-2-azetidinone.]

Step C Preparation of26[1-(t-butyldimethylsilyl)-3-(1-p-nitrobenzylcarbonyldioxyethyl)-4-methyl-4-vinyl-2-azetidinone]##STR26##

Under nitrogen at -78° C. a solution of 25 (0.220 mmole) in 1 ml ofanhydrous tetrahydrofuran is treated with 2.4 M n-butyllithium in hexane(101 μl, 0.242 mmole). To this solution is added, in five minutes, asolution of p-nitrobenzyl chloroformate (52 mg, 0.242 mmole) inanhydrous tetrahydrofuran. After stirring at -78° C. for a period of 55minutes, 10 ml of a saturated aqueous ammonium chloride solution isadded and the product extracted into ethyl acetate. The combined ethylacetate solutions are washed with brine and dried over anhydrousmagnesium sulfate. The drying agent is removed by filtration, and thefiltrate is evaporated in vacuo. Purification by preparative thick-layerchromatography on silica gel developing with 5% ethyl acetate/chloroformgives 26.

Step D Desilylation of 26 to provide27[3-(1-p-nitrobenzylcarbonyldioxyethyl)-4-methyl-4-vinyl-2-azetidinone]##STR27##

A solution of26[1-(t-butyldimethylsilyl)-3-(1-p-nitrobenzylcarbonyldioxyethyl)-4-methyl-4-vinyl-2-azetidinone](0.141 mmole) in 2 ml of 0.5 N HCl/MeOH is stirred at room temperature(25° C.) for a period of 3 hours. The solution is then cooled to 0° C.and neutralized by the addition of 5 ml of 5% aqueous sodiumbicarbonate. The product is extracted into ethyl acetate (10 ml, 2×5ml). The combined ethyl acetate solutions are washed with water (2×5 ml)and 10 ml brine and then dried over anhydrous magnesium sulfate. Thedrying agent is removed by filtration, and the filtrate is evaporated invacuo to give an oil. Preparative thick-layer chromatography of thismaterial on silica gel developing with 10% ethyl acetate/chloroformgives27[3-(1-p-nitrobenzylcarbonyldioxyethyl)-4-methyl-4-vinyl-2-azetidinone].

Step E

Preparation of 14 via 28 by sulfenyl halide addition anddehydrohalogenation ##STR28##

A solution of the N-p-nitroCB^(Z) cysteamine disulfide, 96 mg (0.19mmoles) in 1.5 ml THF (freshly distilled from LiAlH₄) is cooled to -25°C. and treated dropwise with stirring with 0.5 ml of a solution of 135mg Br₂ in sieve dried CCl₄ (2.2 ml final volume; portion added isequivalent to 0.19 mmoles of Br₂). The resultant orange solution isstirred at -20° C. for 5 min. then treated with 54.0 mg of the vinylazetidinone, 27, in 0.5 ml sieve dried CH₂ Cl₂. The color lightens toyellow. The mixture is allowed to come to 0° C. over 5-10 minutes.Examination by tlc (silica 5% MeOH in CH₂ Cl₂ or 20% EtOAc in CH₂ Cl₂)shows a main spot with R_(f) and Ce^(IV+) /H⁺ /heat characteristicsdifferent from either disulfide or starting azetidinone. The reactionmixture is concentrated to 0.5 ml under N₂, streaked directly on two8"×8" 1000μ silica GF plates, and developed with 20% EtOAc in CH₂ Cl₂.The main band under U.V., is scraped off, and extracted with EtOAc togive 28. ##STR29##

Treatment with 1.01 g. AgF(mw=127; 7.95 mmole) of 4.2 mmole of 28 in 34ml. pyridine at room temperature in the dark under N₂ for 1 hr. isfollowed by concentration of the entire reaction mixture under highvacuum and with a water bath at 25°-30° C. to a brown-black residue.After chasing a few times with CHCl₃, the residue is slurried in CH₂ Cl₂and run through a short column of silica gel (eluting with 2% MeOH/CH₂Cl₂) thus removing most of the silver salts. The fractions containingproduct are combined. Chromatography on silica gel (1% MeOH/CH₂ Cl₂)provides the desired product 14. Preparative thin layer chromatographyon silica gel of column fractions which still contained less polarimpurities (2.5% MeOH/CH₂ Cl₂), provides additional product.

Step F

Following the exact procedure described in Example 8, Steps D-K, exceptmaking the indicated substitutions, species 14 of Example 9, Step E, isconverted to I: ##STR30##

EXAMPLE 10 Preparation ofBis(p-Nitrobenzyloxycarbonylaminoethyl)disulfide ##STR31##

Under nitrogen at -20° C., bromine (1.21 ml, 0.022 mmole) is added to asolution of p-nitrobenzyloxycarbonylaminoethanethiol (11.28 g, 0.044mole) in 100 ml of anhydrous tetrahydrofuran. The cooling bath isremoved, and the cold solution is stirred for 15 minutes. The solutionis then diluted with 400 ml ethyl acetate and washed with 200 ml 1 M pH7 phosphate buffer, 200 ml 1 M dibasic potassium phosphate, water (2×200ml, 100 ml) and 200 ml brine. It is dried over anhydrous magnesiumsulfate and filtered. The filtrate is evaporated in vacuo giving ayellow solid residue. This material is chromatographed on silica geleluting with 5% ethyl acetate/chloroform to give 10.5 g of crystallinebis(p-nitrobenzyloxycarbonylaminoethyl)disulfide:

    ______________________________________                                        IR (CH.sub.2 Cl.sub.2)μ:                                                                 3.04NH                                                                        5.96 carbonyl                                                                 6.22, 6.61 nitro                                                NMR (CDCl.sub.3) δ:                                                                   8.24                                                                           d, J = 8.5Hz, ArH                                                            7.54                                                                          5.37, broad s, NH                                                             5.26, s, ArCH.sub.2 O                                                         3.60, q, J = 6Hz and 6Hz, NHCH.sub.2 CH.sub.2                                 2.86, t, J = 6Hz, NHCH.sub.2 CH.sub.2 S                         ______________________________________                                    

EXAMPLE 11

Following the procedure of the foregoing Examples and text exceptsubstituting the appropriately substituted acyloxybutadiene for theillustrated methyl substituted acyloxybutadiene starting material, thereis obtained the following representative species of the presentinvention. ##STR32##

For every entry in Table I, the butadiene reagent is given by thegeneral formula: ##STR33## wherein R is defined in Table I.

                  TABLE I                                                         ______________________________________                                        Com-                                                                          pound R                   Reagents, Remarks                                   ______________________________________                                        1.    CH.sub.2 CH.sub.3                                                              ##STR34##                                                                     ##STR35##                                                                     ##STR36##                                                                     ##STR37##                                                                     ##STR38##                                                                     ##STR39##                                                                     ##STR40##                                                                     ##STR41##                                                              10.                                                                                  ##STR42##                                                                     ##STR43##                                                                     ##STR44##                                                                     ##STR45##                                                                     ##STR46##                                                                     ##STR47##                                                                     ##STR48##                                                                     ##STR49##                                                              18.   CF.sub.3                                                                       ##STR50##                                                              20.                                                                                  ##STR51##                                                              ______________________________________                                    

EXAMPLE 12 Preparation of Pharmaceutical Compositions

One such unit dosage form consists in mixing 120 mg of I: ##STR52## with20 mg of lactose and 5 mg of magnesium stearate and placing the 145 mgmixture into a No. 3 gelatin capsule. Similarly, by employing more ofthe active ingredient and less lactose, other dosage forms can be put upin No. 3 gelatin capsules and should it be necessary to mix more than145 mg of ingredients together, larger capsules such as compressedtablets and pills can also be prepared. The following examples areillustrative of the preparation of pharmaceutical formulations:

    ______________________________________                                        TABLET           PER TABLET                                                   ______________________________________                                        I                125 mg.                                                      Cornstarch, U.S.P.                                                                              6 mg.                                                       Dicalcium Phosphate                                                                            192 mg.                                                      Lactose, U.S.P.  190 mg.                                                      Magnesium Stearate                                                                             Balance/800 mg.                                              ______________________________________                                    

The active ingredient is blended with the dicalcium phosphate, lactoseand about half of the cornstarch. The mixture is then granulated with15% cornstarch paste (6 mg) and rough-screened. It is dried at 45° C.and screened again through No. 16 screens. The balance of the cornstarchand magnesium stearate is added and the mixture is compressed intotablets, approximately 0.5 inch in diameter each weighing 800 mg.

    ______________________________________                                                             PER TABLET                                               ______________________________________                                        PARENTERAL SOLUTION                                                           Ampoule:                                                                      I                          500 mg.                                            Diluent: Sterile water for injection                                                                      2 cc.                                             OPTHALMIC SOLUTION                                                            I                          100 mg.                                            Hydroxypropylmethyl cellulose                                                                             5 mg.                                             Sterile water        to     1 ml.                                             OTIC SOLUTION                                                                 I                          100 mg.                                            Benzalkonium chloride      0.1 mg.                                            Sterile water        to     1 ml.                                             TOPICAL OINTMENT                                                              I                          100 mg.                                            Polyethylene Glycol 4000 U.S.P.                                                                          400 mg.                                            Polyethylene Glycol 400 U.S.P.                                                                           1.0 gram.                                          ______________________________________                                    

What is claimed is:
 1. A compound of the structure ##STR53## and thepharmaceutically acceptable salts thereof; wherein R is a memberselected from the group consisting of loweralkyl, cycloloweralkyl,cycloloweralkylloweralkyl, benzyl, phenyl, chlorophenyl, bromophenyl,fluorophenyl, methoxyphenyl, loweralkylphenyl and trifluoromethyl.
 2. Acompound according to claim 1 wherein R is a member selected from thegroup consisting of methyl, ethyl, isopropyl, phenyl, p-methylphenyl,benzyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl andtrifluoromethyl.
 3. An antibiotic pharmaceutical composition comprisinga therapeutically effective amount of a compound according to claim 1and a pharmaceutical carrier therefor.
 4. A compound having thestructure: ##STR54## and its pharmaceutically acceptable salts.